Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which kills 2 million people per year and infects nearly one-third of the world's population. Recent outbreaks of multi-drug resistant Mtb strains and the deadly synergy of TB and AIDS stress the need for new and more effective treatments for TB. We propose to characterize the signal transduction mechanism of the PhoR sensor histidine kinase and its cognate response regulator PhoP from Mtb. This two-component system is essential for virulence and intracellular growth of Mtb. Global gene profiling studies indicate that at least 44 genes are positively regulated and 70 genes are negatively regulated by PhoP-PhoR. Therefore, this signaling system must play an important role in adaptation of the pathogen to its intracellular environments, making the PhoP-PhoR system a potential target for novel anti-tuberculosis drugs. However, no structural data is available for this two-component system, and the mechanism of signal transduction is unknown. The specific aims of this proposal are (1) to determine the crystal structure of PhoP;(2) to define the mechanism of DMA sequence recognition by PhoP;(3) to investigate the role of each domain of PhoR in dimer formation and regulation of the protein activity;and (4) to determine crystal structures of truncated domains and the full-length PhoR protein. Toward achieving these aims, we have (i) determined the structure of the C-terminal domain of PhoP and obtained crystals of full-length PhoP;(ii) identified several promoters that bind PhoP and mapped the PhoP binding sites, from which we will identify optimal DMA sequences for structural determination of PhoP-DNA complexes;and (iii) purified full-length PhoR and prepared expression constructs for producing various truncated domains of PhoR. We will use a divide-and-conquer strategy to study the isolated domains in parallel with the full-length proteins. Structural and functional information of separate domains will lead to success in determining the structure of the full-length protein. The structure of intact PhoR will provide the first integral membrane protein structure of a large group of sensor histidine kinases. Results from this research will lead to a detailed molecular mechanism of signal transduction by PhoP-PhoR and thus a better understanding of how the pathogen adapts to intracellular environments. High resolution crystal structures will also provide a basis for the design of better therapeutics.